Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A2 activities.
Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A2 (PLA2) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/microg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/microg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/microg). PLA2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.
Wang
J
J
Department of Comparative Medicine, Wake Forest University, School of Medicine, Winston-Salem, NC 27157, USA.
DeLozier
J A
JA
Gebre
A K
AK
Dolphin
P J
PJ
Parks
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002788:Cholesterol Esters / Q000378:metabolism*; D004951:Esterification; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014162:Transfection
D002788:Cholesterol Esters; D018698:Glutamic Acid; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D010741:Phospholipases A; D054467:Phospholipases A2